Evaluation of methods for isolation of DNA from slowly and rapidly growing mycobacteria.
Mycobacteria generally have thick cell walls and contain large amounts of lipid, making them resistant to DNA extraction. Five methods, namely, extensive enzymic digestion method (M1), 2-min mechanical glass-bead disruption method (M2), thermal shock method (M3), modified conventional enzymic digestion method (M4), and manual disruption with modified conventional enzymic digestion method (M5), were used to compare their effectiveness and simplicity in extracting DNA from slowly growing mycobacteria (Mycobacterium leprae, M. lepraemurium and M. bovis BCG), and a rapidly growing mycobacterium (M. phlei). The highest DNA yield was obtained by M2 from M. lepraemurium which produced 2.82 micrograms DNA/mg wet weight of cells, representing a theoretical yield of 78%. M3 gave the lowest DNA yield; 0.01 microgram DNA/mg wet weight of cells of M. lepraemurium was obtained. M4, in which proteinase K was used, is more effective than M1, in which subtilisin and pronase were used. M5 yielded a higher amount of DNA, but it required more manipulations to extract DNA as compared to M4. Extraction of DNA of M. leprae from nude mice is more difficult than that of M. leprae from armadillos by all of the methods used. These results suggest that the biosynthetic capabilities of these two forms of M. leprae may vary, depending on their cultural conditions and/or strain differences. Our results have shown that both M2 and M4 are the simplest, most effective and time-saving methods which are suitable for every routine laboratory to extract DNA from slowly and rapidly growing mycobacteria.
Zhang
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ZQ
Ishaque
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D000818:Animals; D001135:Armadillos; D001431:Bacteriological Techniques; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004798:Enzymes / Q000378:metabolism; D005821:Genetic Techniques; D018869:Heat-Shock Response; D051379:Mice; D008809:Mice, Inbred C3H; D008819:Mice, Nude; D008967:Molecular Biology / Q000379:methods*; D009161:Mycobacterium / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D009163:Mycobacterium bovis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D009166:Mycobacterium leprae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D009167:Mycobacterium lepraemurium / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D009168:Mycobacterium phlei / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
D004269:DNA, Bacterial; D004798:Enzymes; D004262:DNA Restriction Enzymes